This invention generally relates to luciferase enzymes that produce luminescence, like that from fireflies. More particularly, the invention concerns mutant luciferases of beetles. The mutant luciferases of the invention are made by genetic engineering, do not occur in nature, and, in each case, include modifications which cause a change in color in the luminescence that is produced. The luciferases of the invention can be used, like their naturally occurring counterparts, to provide luminescent signals in tests or assays for various substances or phenomena.
The use of reporter molecules or labels to qualitatively or quantitatively monitor molecular events is well established. They are found in assays for medical diagnosis, for the detection of toxins and other substances in industrial environments, and for basic and applied research in biology, biomedicine, and biochemistry. Such assays include immunoassays, nucleic acid probe hybridization assays, and assays in which a reporter enzyme or other protein is produced by expression under control of a particular promoter. Reporter molecules, or labels in such assay systems, have included radioactive isotopes, fluorescent agents, enzymes and chemiluminescent agents.
Included in the assay system employing chemiluminescence to monitor or measure events of interest are assays which measure the activity of a bioluminescent enzyme, luciferase.
Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus Pyrophorus and the fireflies of the genera Photinus, Photuris, and Luciola. In many of these organisms, enzymes catalyze monooxygenations and utilize the resulting free energy to excite a molecule to a high energy state. Visible light is emitted when the excited molecule spontaneously returns to the ground state. This emitted light is called xe2x80x9cbioluminescence.xe2x80x9d Hereinafter it may also be referred to simply as xe2x80x9cluminescence.xe2x80x9d
The limited occurrence of natural bioluminescence is an advantage of using luciferase enzymes as reporter groups to monitor molecular events. Because natural bioluminescence is so rare, it is unlikely that light production from other biological processes will obscure the activity of a luciferase introduced into a biological system. Therefore, even in a complex environment, light detection will provide a clear indication of luciferase activity.
Luciferases possess additional features which render them particularly useful as reporter molecules for biosensing (using a reporter system to reveal properties of a biological system). Signal transduction in biosensors (sensors which comprise a bilogical component) generally involves a two step process: signal generation through a biological component, and signal transduction and amplification through an electrical component. Signal generation is typically achieved through binding or catalysis. Conversion of these biochemical events into an electrical signal is typically based on electrochemical or caloric detection methods, which are limited by the free energy change of the biochemical reactions. For most reactions this is less than the energy of hydrolysis for two molecules of ATP, or about 70 kJ/mole. However, the luminescence elicited by luciferases carries a much higher energy content. Photons emitted from the reaction catalyzed by firefly luciferase (560 nm) have 214 Kj/einstein. Furthermore, the reaction catalyzed by luciferase is one of the most efficient bioluminescent reactions known, having a quantum yield of nearly 0.9. This enzyme is therefore an extremely efficient transducer of chemical energy.
Since the earliest studies, beetle luciferases, particularly that from the common North American firefly species Photinus pyralis, have served as paradigms for understanding of bioluminescence . The fundamental knowledge and applications of luciferase have been based on a single enzyme, called xe2x80x9cfirefly luciferase,xe2x80x9d derived from Photinus Rvralis. However, there are roughly 1800 species of luminous beetles worldwide. Thus, the luciferase of Photinus pyralis is a single example of a large and diverse group of beetle luciferases. It is known that all beetle luciferases catalyze a reaction of the same substrate, a polyheterocyclic organic acid, D-(xe2x88x92)-2-(6xe2x80x2-hydroxy-2xe2x80x2-benzothiazolyl)-Axcex942-thiazoline -4-carboxylic acid (hereinafter referred to as xe2x80x9cluciferinxe2x80x9d, unless otherwise indicated), which is converted to a high energy molecule. It is likely that the catalyzed reaction entails the same mechanism in each case.
The general scheme involved in the mechanism of beetle bioluminescence appears to be one by which the production of light takes place after the oxidative decarboxylation of the luciferin, through interaction of the oxidized luciferin with the enzyme. The color of the light apparently is determined by the spatial organization of the enzyme""s amino acids which interact with the oxidized luciferin.
The luciferase-catalyzed reaction which yields bioluminescence (hereinafter referred to simply as xe2x80x9cthe luciferase-luciferin reactionxe2x80x9d) has been described as a two-step process involving luciferin, adenosine triphosphate (ATP), and molecular oxygen. In the initial reaction, the luciferin and ATP react to form luciferyl adenylate with the elimination of inorganic pyro-phosphate, as indicated in the following reaction:
E+LH2+ATPxe2x86x92Exc2x7LH-AMP+PPi
where E is the luciferase, LH2 is luciferin, and PPi is pyrophosphate. The luciferyl adenylate, LH2-AMP, remains tightly bound to the catalytic site of luciferase. When this form of the enzyme is exposed to molecular oxygen, the enzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin (L=0) in an electronically excited state. The excited oxidized luciferin emits light on returning to the ground state as indicated in the following reaction: 
One quantum of light is emitted for each molecule of luciferin oxidized. The electronically excited state of the oxidized luciferin is a characteristic state of the luciferase-luciferin reaction of a beetle luciferase; the color (and, therefore, the energy) of the light emitted upon return of the oxidized luciferin to the ground state is determined by the enzyme, as evidenced by the fact that various species of beetles having the same luciferin emit differently colored light.
Luciferases have been isolated directly from various sources. The cDNAs encoding luciferases of various beetle species have been reported. (See de Wet et al., Molec. Cell. Biol 7, 725-737 (1987); Masuda et al., Gene 77, 265-270 (1989); Wood et al., Science 244, 700-702 (1989)). With the cDNA encoding a beetle luciferase in hand, it is entirely straightforward for the skilled to prepare large amounts of the luciferase by isolation from bacteria (e.g., E. coli), yeast, mammalian cells in culture, or the like, which have been transformed to express the cDNA. Alternatively, the cDNA, under control of an appropriate promoter and other signals for controlling expression, can be used in such a cell to provide luciferase, and ultimately bioluminescence catalyzed thereby, as a signal to indicate activity of the promoter. The activity of the promoter may, in turn, reflect another factor that is sought to be monitored, such as the concentration of a substance that induces or represses the activity of the promoter. Various cell-free systems, that have recently become available to make proteins from nucleic acids encoding them, can also be used to make beetle luciferases.
Further, the availability of cDNAS encoding beetle luciferases and the ability to rapidly screen for CDNAS that encode enzymes which catalyze the luciferase-luciferin reaction (see de Wet et al., supra and Wood et al., supra) also allow the skilled to prepare, and obtain in large amounts, other luciferases that retain activity in catalyzing production of bioluminescence through the luciferase-luciferin reaction. These other luciferases can also be prepared, and the cDNAs that encode them can also be used, as indicated in the previous paragraph. In the present disclosure, the term xe2x80x9cbeetle luciferasexe2x80x9d or xe2x80x9cluciferasexe2x80x9d means an enzyme that is capable of catalyzing the oxidation of luciferin to yield bioluminescence, as outlined above.
The ready availability of cDNAS encoding beetle luciferases makes possible the use of the luciferases as reporters in assays employed to signal, monitor or measure genetic events associated with transcription and translation, by coupling expression of such a cDNA, and consequently production of the enzyme, to such genetic events.
Firefly luciferase has been widely used to detect promoter activity in eucaryotes. Though this enzyme has also been used in procaryotes, the utility of firefly luciferase as genetic reporter in bacteria is not commonly recognized. As genetic reporters, beetle luciferases are particularly useful since they are monomeric products of a single gene. In addition, no post-translational modifications are required for enzymatic activity, and the enzyme contains no prosthetic groups, bound cofactors, or disulfide bonds. Luminescence from E.coli containing the gene for firefly luciferase can be triggered by adding the substrate luciferin to the growth medium. Luciferin readily penetrates biological membranes and cannot be used as a carbon or nitrogen source by E.coli. The other substrates required for the bioluminescent reaction, oxygen and ATP, are available within living cells. However, measurable variations in luminescence color from luciferases would be needed for systems which utilize two or more different luciferases as reporters (signal geneators).
Clones of different beetle luciferases, particularly of a single genus or species, can be utilized together in bioluminescent reporter systems. Expression in exogenous hosts should differ little between these luciferases because of their close sequence similarity. Thus, in particular, the click beetle luciferases may provide a multiple reporter system that can allow the activity of two or more different promoters to be monitored within a single host, or for different populations of cells to be observed simultaneously. The ability to distinguish each of the luciferases in a mixture, however, is limited by the width of their emissions spectra.
One of the most spectacular examples of luminescence color variation occurs in Pyrophorus plagiophthalamus, a large click beetle indigenous to the Caribbean. This beetle has two sets of light organs, a pair on the dorsal surface of the prothorax, and a single organ in a ventral cleft of the abdomen. Four different luciferase clones have been isolated from the ventral organ. The luciferin-luciferase reactions catalyzed by these enzymes produces light that ranges from green to orange.
Spectral data from the luciferase-luciferin reaction catalyzed by these four luciferases show four overlapping peaks of nearly even spacing, emitting green (peak intensity: 546 nanometers), yellow-green (peak intensity: 560 nanometers), yellow (peak intensity: 578 nanometers) and orange (peak intensity: 593 nanometers) light. The respective proteins are named LucPplGR, LucPplYG, LucPplYE and LucPpOR. Though the wavelengths of peak intensity of the light emitted by these luciferases range over nearly 50 nm, there is still considerable overlap among the spectra, even those with peaks at 546 and 593 nm. Increasing the difference in wavelength of peak intensity would thus be useful to obtain greater measurement precision in systems using two or more luciferases.
The amino acid sequences of the four luciferases from the ventral organ are highly similar. Comparisons of the sequences show them to be 95 to 99% identical.
It would be desirable to enhance the utility of beetle luciferases for use in systems using multiple reporters to effect mutations in luciferase-encoding cDNAs to produce mutant luciferases which, in the luciferase-luciferin reaction, produce light with differences between wavelengths of peak intensity that are greater than those available using currently available luciferases.
Beetle luciferases are particularly suited for producing these mutant luciferases since color variation is a direct result of changes in the amino acid sequence.
Mutant luciferases of fireflies of genus Luciola are known in the art. Kajiyama et al., U.S. Pat. Nos. 5,219,737 and 5,229,285.
In using luciferase expression in eukaryotic cells for biosensing, it would be desirable to reduce transport of the luciferase to peroxisomes. Sommer et al., Mol. Biol. Cell 3, 749-759 (1992), have described mutations in the three carboxy-terminal amino acids of P. pyralis luciferase that significantly reduce peroxisome-targeting of the enzyme.
The sequences of cDNAs enoding various beetle luciferases, and the amino acid sequences deduced from the cDNA sequences, are known, as indicated in Table I.
The CDNA and amino acid sequences of LucPplGR, the green-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, are shown in SEQ ID NO:1.
The amino acid sequence of LucPplGR, the green-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, is shown in SEQ ID NO:2.
The amino acid and cDNA sequences of LucPplYG, the yellow-green-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, are shown in SEQ ID NO:3.
The amino acid sequence of LucPplYG, the yellow-green-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, is shown in SEQ ID NO:4.
The amino acid and cDNA sequences of LucPplYE, the yellow-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, are shown in SEQ ID NO:5.
The amino acid sequence of LucPplYE, the yellow-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, is shown in SEQ ID NO:6.
The cDNA and amino acid sequences of LucPplOR, the orange-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, are shown in SEQ ID NO:7.
The amino acid sequence of LucPplOR, the orange-emitting luciferase of the elaterid beetle Pyrophorus plagiophthalamus, is shown in SEQ ID NO:8.
The cDNA and amino acid sequences of the luciferase of Photinus pyralis are shown in SEQ ID NO:9.
The amino acid sequence of the luciferase of Photinus pyralis is shown in SEQ ID NO:10.
The cDNA and amino acid sequences of the luciferase of Luciola cruciata are shown in SEQ ID NO:11.
The amino acid sequence of the luciferase the of Luciola cruciata is shown in SEQ ID NO:12.
The CDNA and amino acid sequences of the luciferase of Luciola lateralis are shown in SEQ ID NO:13.
The amino acid sequence of the luciferase of Luciola lateralis is shown in SEQ ID NO:14.
The CDNA and amino acid sequences of the luciferase of Luciola mingrelica are shown in SEQ ID NO:15.
The amino acid sequence of the luciferase of Luciola mingrelica is shown in SEQ ID NO:16.
The present invention provides mutant luciferases of beetles and DNAs which encode the mutant luciferases. Preferably, the mutant luciferases produce a light of different color from that of the corresponding wild-type luciferase and preferably this difference in color is such that the wavelength of peak intensity of the luminescence of the mutant differs by at least 1 nm from that of the wild-type enzyme.
The mutant luciferases of the invention differ from the corresponding wild-type enzymes by one or more, but typically fewer than three, amino acid substitutions. The luciferases of the invention may also entail changes in one or more of the three carboxy-terminal amino acids to reduce peroxisome targeting.
In one surprising aspect of the invention, it has been discovered that combining in a single mutant two amino acid substitions, each of which, by itself, occasions a change in color (shift in wavelength of peak intensity) of bioluminescence, causes the mutant to have a shift in wavelength of peak intensity that is greater than either shift caused by the single amino acid substitutions.
cDNAs encoding the mutant luciferases of the invention may be obtained straightforwardly by any standard, site-directed mutagenesis procedure carried out with a cDNA encoding the corresponding wild-type enzyme or another mutant. The mutant luciferases of the invention can be made by standard procedures for expressing the cDNAs which encode them in prokaryotic or eukaryotic cells.
A fuller appreciation of the invention will be gained upon examination of the following detailed description of the invention.